A calcium-binding photoprotein is one of the proteins responsible for bioluminescence. This photoprotein instantaneously emits a flash of light upon specific interaction with Ca2+. The calcium-binding photoprotein is a complex of a protein having the catalytic function of oxygenation and the peroxide of a luciferin as a luminescence substrate. In the calcium-binding photoprotein, the protein having the catalytic function of oxygenation is called an apoprotein (e.g., apoaequorin). The peroxide of a luciferin is 2-hydroperoxycoelenterazine. Such known calcium-binding photoproteins are those derived from coelenterates, specifically including aequorin, clytin-I, clytin-II, mitrocomin, obelin, etc.
Among them, aequorin is a photoprotein isolated from the luminous jellyfish Aequorea aequorea (1: Shimomura, In: Bioluminescence, Chemical Principles and Methods, (2006) pp 90-158, World Scientific Pub. Co.; 2: Shimomura et al., (1962) J. Cell. Comp. Physiol. 59, pp 223-240). This aequorin is a non-covalent complex of apoaequorin (21.4 kDa) and a hydroperoxide of coelenterazine (3: Head et al., (2000) Nature, 405 372-376). Apoaequorin is a single polypeptide composed of 189 amino acid residues with 3 EF hand motifs characteristic of Ca2+-binding site (4: Inouye et al., (1985) Proc. Natl. Acad. Sci. USA. 82, 3154-3158). In the presence of Ca2+, acquorin emits blue light (λmax=˜460 nm) by an intramolecular reaction and decomposes itself into apoaequorin, coelenteramide and CO2 (5: Shimomura & Johnson (1972) Biochemistry 11, 1602-1608; 6: Shimomura & Johnson (1973) Tetrahedron Let. 2963-2966). The complex of Ca2−-bound apoaequorin with coelenteramide obtained by this decomposition is known as blue fluorescent protein (BFP) (7: Shimomura & Johnson (1975) Nature 256, 236-238).
The fluorescence emission spectrum of BFP is the same as bioluminescence spectrum of aequorin (8: Shimomura, Biochem. J. 306 (1995) 537-543; 9: Inouye, FEBS Lett. 577 (2004) 105-110). Recombinant apoaequorin prepared from E. coli can be regenerated into aequorin by incubation with coelenterazine and molecular oxygen in the presence of EDTA and a reducing agent (10: Inouye et al., (1986) Biochemistry 25: 8425-8429; 11: Inouye et al., (1989) J. Biochem., 105, 473-477). Recombinant aequorin is highly purified (12: Shimomura & Inouye (1999) Protein Express. Purif 16, 91-95). Recombinant aequorin has identical luminescence properties to those of native aequorin (13: Shimomura et al., (1990) Biochem. J. 270 309-312). The crystal structures of aequorin and semi-synthetic aequorin were determined (3: Head et al., Nature, 405 (2000) 372-376; 14: Toma et al., (2005) Protein Science 14:409416) and the Mg2+ binding properties of aequorin to EF hand motifs were also examined by NMR spectroscopy (15: Ohashi et al., (2005) J. Biochem. 138: 613-620).
Recently, BFP was quantitatively prepared from the purified recombinant aequorin (9: Inouye, FEBS Lett. 577 (2004) 105-110; 16: Inouye & Sasaki, FEBS Lett. 580 (2006) 1977-1982). BFP was found to have a substantial luminescence activity, catalyzing the oxidation of coelenterazine like a luciferase. The luminescence intensity of BFP is about 10 times higher than that of Ca2+-bound apoaequorin (9: Inouye, FEBS Lett. 577 (2004) 105-110). That is, BFP is a new bifunctional protein having both fluorescence and luciferase activities. Furthermore, by treatment with EDTA, BFP is converted into a green fluorescent protein (gFP) showing the maximum fluorescence emission peak at about 470 nm. gFP is a non-covalent complex of apoaequorin and coelenteramide and may reconstitute aequorin by incubation with coelenterazine at 25° C. in the absence of reducing reagents (9: Inouye, FEBS Lett. 577 (2004) 105-110). By incubation of various coelenterazine analogs with BFP or gFP in the presence of EDTA and dithiothreitol (DTT), semi-synthetic aequorin may also be prepared (16: Inouye et al. & Sasaki, FEBS Lett. 580 (2006) 1977-1982). Moreover, the luminescence activity of BFP as a luciferase is stimulated by adding imidazole at concentrations of 30 to 300 mM using coelenterazine and its analog as substrates (17: Inouye & Sasaki (2007) Biochem. Biophys. Res. Commun. 354: 650-655). However, the protein catalytic domains or amino acid residues for the oxygen addition to coelenterazine, which are important basic information for developing the applications of BFP and gFP, remain unknown. In solving these problems and developing the applications, it is required to easily prepare BFP and gFP of several ten milligrams. Put otherwise, it is essential to establish a process for preparing highly purified gFP from apoaequorin and coelenteramide and converting gFP into BFP.
Turning now to the production of coelenteramide, which is the starting material for producing BFP and gFP, there is known a process for producing coelenteramide, which comprises reacting the compound shown by formula below:
with p-hydroxyphenylacetic acid shown by formula below:
(6: Shimomura & Johnson, Tetrahedron Lett. (1973) 2963-2966). According to this process, the yield of coelenteramide is approximately 50%.    1. Shimomura, In: Bioluminescence, Chemical principles and methods (2006) pp 90-158, World Scientific Pub. Co.    2. Shimomura et al., (1962) J. Cell. Comp. Physiol. 59, pp 223-240    3. Head et al., (2000) Nature, 405, 372-376    4. Inouye et al., (1985) Proc. Natl. Acad. Sci. USA. 82, 3154-3158    5. Shimomura & Johnson (1972) Biochemistry 11, 1602-1608    6. Shimomura & Johnson (1973) Tetrahedron Len. 2963-2966    7. Shimomura & Johnson (1975) Nature 256, 236-238    8. Shimomura, (1995) Biochem. J. 306, 537-543    9. Inouye, (2004) FEBS Lett. 577, 105-110    10. Inouye et al., (1986) Biochemistry 25, 8425-8429    11. Inouye et al., (1989) J. Biochem., 105 473-477    12. Shimomura & Inouye (1999) Protein Express. Purif 16, 91-95    13. Shimomura et al., (1990) Biochem. J. 270, 309-312    14. Toma et al., (2005) Protein Science 14, 409-416    15. Ohashi et al., (2005) J. Biochem. 138, 613-620    16. Inouye & Sasaki, (2006) FEBS Lett. 580, 1977-1982    17. Inouye & Sasaki, (2007) Biochem. Biophys. Res. Commun. 354, 650-655